Certain cells of the immune system produce proteins called antibodies or immunoglobulins (“Ig”) in response to the presence of foreign proteins in the body, such as bacterial or viral proteins. Antibodies bind and neutralize foreign proteins in the body.
Antibodies generally bind their target protein antigens tightly and specifically, making them potentially useful therapeutics for treating a wide range of diseases characterized by altered protein expression. Many protein targets suitable for antibody-mediated disease therapy have been identified using non-human antibody molecules. For many therapeutic applications, however, the efficacy and safety of non-human antibodies is compromised because non-human Ig molecules are themselves immunogenic (i.e., capable of inducing an immune response). Thus, before antibodies can be approved for therapeutic use, they normally must be modified to reduce or eliminate their immunogenicity. Antibody Humaneering™ produces antibodies modified to reduce immunogenicity while retaining the ability to specifically bind their target antigen.
The present application describes the “humaneering” of a murine monoclonal antibody that binds factor B and selectively blocks the alternative complement pathway. The alternative complement pathway is usually activated by bacteria, parasites, viruses or fungi, although IgA antibodies and certain Ig light chains have also been reported to activate the pathway. Alternative pathway activation is initiated when circulating factor B binds to activated C3 (either C3b or C3H2O). This complex is then cleaved by circulating factor D to yield an enzymatically active fragment, either C3bBb or C3(H2O)Bb. These two enzymes can cleave circulating C3 generating C3b, which drives inflammation and also further amplifies the activation process, generating a positive feedback loop. Factor B is required to enable activation of the alternative pathway.
Recent studies have shown that the alternative pathway of complement plays an important role in the pathogenesis of several animal models of disease. Complement activation within the kidney after ischemia/reperfusion injury is mediated almost exclusively by the alternative pathway and the alternative pathway plays a critical role in the development of arthritis. Perhaps most surprisingly, mice deficient in the alternative pathway have been demonstrated to be protected from nephritis in the MRL/lpr model of lupus nephritis and from anti-phospholipid mediated fetal loss, disease models that would traditionally have been assumed to be mediated by the classical complement pathway.
The murine anti-factor B antibody from which the humaneered variants described herein were derived was produced by injecting factor B deficient mice (“fB−/−”) with a fusion protein comprising the second and third short consensus repeat (“SCR”) domains of factor B fused to an immunoglobulin. The mice were then screened for antibodies to factor B. Spleen cells from an injected mouse producing anti-factor B antibodies were fused to myeloma cells according to standard procedures known in the art. One of the resulting hybridoma cells, number 1379, produced an IgG1 antibody (“mAb 1379”) that completely inhibits activation of the alternative complement pathway in vitro and in vivo. Antigen-binding Fab′ fragments of mAb 1379 also completely inhibit activation of the alternative complement pathway. The hybridoma cell line that produces mAb 1379 has been deposited with the American Type Culture Collection (“ATCC”) under Deposit No. PTA-6230.
Epitope mapping showed that mAb 1379 binds to factor B within the third SCR domain. Further experiments demonstrated that mAb 1379 inhibits alternative complement activation by preventing formation of the C3bBb complex. Finally, mAB 1379 binds an epitope conserved across multiple mammalian species, as shown by its ability to inhibit alternative complement activation in serum from a number of different species, including mice, rats, humans, baboons, rhesus monkeys, cynomolgous monkeys, pigs, rabbits, and horses. The production and characterization of anti-factor B antibody mAb 1379 is described in greater detail in U.S. Patent Publication No. US 2005/0260198 A1, which is incorporated herein by reference.
All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.